It is so far known that cells of a glycosaminoglycan (GAG) degrading enzyme producer microorganism or culture media obtained upon cultivation of such microbial producer contain GAG degrading enzymes such as lyase, glycuronidase, sulfatase and other enzyme species.
An analytical method is also known for GAG sugar chain structure determination using lyase, glycuronidase, sulfatase and other enzyme species isolated from cells of the glycosaminoglycan degrading enzyme producer or culture media obtained upon cultivation of such producer (cf. Federation Proceedings, 36, 43 (1977); and The Journal of Biological Chemistry, 243, 1536 (1968)).
For the separation and purication of these enzymes from cell- and/or culture media-derived liquid preparations containing the same, there have been proposed a column chromatographic method using hydroxyapatite, a method using ion exchange chromatography and gel filtration combinedly, and a chromatographic method which utilizes the affinities of lyases, glycuronidases, sulfatases, etc. for immobilized heparin or dermatan sulfate (Carbohydrate Research, 70, 295, 1979; ibid., 88, 291, 1981), among others.
However, the column chromatographic method of separation and purification using hydroxyapatite is still disadvantageous in that it is difficult to separate the above-mentioned enzymes from one another, although the method is excellent in that impurities other than those enzymes can be removed efficiently therefrom.
The method using ion exchange chromatography and gel filtration combinedly can hardly separate Flavobacterium haparinum-derived heparitinase and sulfatase from each other or heparitinase and glycuronidase from each other. The chromatographic method using immobilized heparin or dermatan sulfate is also disadvantageous in that the column cannot be used repeatedly and in that the mutual separation of enzymes is unsatisfactory.
If enzymes separated and purified by such prior art methods are used for GAG sugar chain structure determination, serious errors may be made in GAG cleavage site determination or in sulfate group cleavage site determination since such enzymes are not sufficiently pure and each is not a single enzyme.